BMJ Open · For peer review only Expression of prostaglandin E receptor subtype EP4 in conjunctival...

For peer review only

Expression of prostaglandin E receptor subtype EP4 in conjunctival epithelium of patients with ocular surface

disorders

Journal: BMJ Open

Manuscript ID: bmjopen-2012-001330

Article Type: Research

Date Submitted by the Author: 30-Apr-2012

Complete List of Authors: Ueta, Mayumi; Kyoto Prefectural University of Medicine, Department of Ophthalmology Sotozono, Chie; Kyoto Prefectural Univ of Med, Ophthalmology

Yamada, Keiko; Kyoto Prefectural University of Medicine, Department of Ophthalmology Yokoi, Norihiko; Kyoto Prefectural University of Medicine, Department of Ophthalmology Inatomi, Tsutomu; Kyoto Prefectural University of Medicine, Department of Ophthalmology Kinoshita, Shigeru; Kyoto Prefectural University of Medicine, Department of Ophthalmology

<b>Primary Subject Heading</b>:

Ophthalmology

Secondary Subject Heading: Ophthalmology

Keywords: OPHTHALMOLOGY, Corneal and external diseases < OPHTHALMOLOGY,

Histology < BASIC SCIENCES

For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml

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1

Expression of prostaglandin E receptor subtype EP4 in conjunctival epithelium of

patients with ocular surface disorders: case-control study

Mayumi Ueta, MD, PhD,a,b

Chie Sotozono, MD, PhD,a Keiko Yamada, MD,

a

Norihiko Yokoi, MD, PhD,a Tsutomu Inatomi, MD, PhD,

a Shigeru Kinoshita, MD, PhD

a

aDepartment of Ophthalmology, Kyoto Prefectural University of Medicine,

Kyoto,

Japan

bResearch Center for Inflammation and Regenerative Medicine, Faculty of Life and

Medical Sciences, Doshisha University, Kyoto, Japan

Corresponding author:

Mayumi Ueta, MD, PhD

Department of Ophthalmology

Kyoto Prefectural University of Medicine

465 Kajiicho, Hirokoji

Kawaramachi, Kamigyoku

Kyoto 602-0841, Japan

Telephone: 81-75-251-5578

Fax: 81-75-251-5663

e-mail: [email protected]

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Key words

Prostaglandin E receptor subtype EP4, human conjunctival epithelium, chemical eye

burn, Mooren’s ulcer

Abbreviations

EP4: Prostaglandin E receptor subtype EP4

SJS: Stevens-Johnson syndrome

TEN: Toxic epidermal necrolysis

GVHD: graft versus host disease

OCP: ocular cicatricial pemphigoid

RT-PCR: Reverse transcription polymerase chain reaction

PG: Prostaglandin

1) substantial contributions to conception and design, acquisition of data, or analysis and

interpretation of data; Mayumi Ueta, Chie Sotozono, Keiko Yamada, Norihiko Yokoi,

Tsutomu Inatomi, Shigeru Kinoshita

2) drafting the article or revising it critically for important intellectual content; Mayumi

Ueta, Chie Sotozono, Keiko Yamada, Norihiko Yokoi, Tsutomu Inatomi, Shigeru

Kinoshita

3) final approval of the version to be published; Mayumi Ueta, Chie Sotozono, Keiko

Yamada, Norihiko Yokoi, Tsutomu Inatomi, Shigeru Kinoshita

There is no additional data available.

Competing interest statement: none

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Abstract

Objectives: To examine the expression of EP4 in conjunctival epithelium of patients with

various ocular surface disorders.

Design: case-control study

Setting & Participants: Conjunctival tissues were obtained from patients undergoing

surgical reconstruction of the ocular surface due to chemical eye burns, sub-acute and

chronic stage SJS/TEN, chronic stage ocular cicatricial pemphigoid (OCP) or severe

graft versus host disease (GVHD), and from patients with Mooren's ulcers treated by

resection of the inflammatory conjunctiva. We performed immunohistological analysis of

EP4 and quantitative RT-PCR analysis of conjunctival tissue sections to confirm the

down-regulation of EP4 mRNA expression in patients with SJS/TEN and OCP.

Primary and secondary outcome measures: Expression of EP4 protein by

immunohistological methods and expression of EP4 mRNA.

Results: EP4 protein was detected in conjunctival epithelium from patients with

chemical eye burn and in control conjunctival epithelium from patients with

conjunctivochalasis. The expression of EP4 protein varied in conjunctival epithelium

from patients with Mooren’s ulcer. We did not detect EP4 immunoreactivity in

conjunctival epithelium from patients with subacute SJS/TEN, severe GVHD, chronic

SJS/TEN, or OCP.

Conclusions: The strong down-regulation of EP4 expression in conjunctival epithelium

from patients with OCP or SJS/TEN may be attributable to ocular surface inflammation.

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Introduction

The prostanoids PGD2, PGE2, PGF2α, PGI2, and TXA2 are lipid mediators that

form in response to various stimuli. They are released extracellularly immediately after

their synthesis and they act by binding to a G-protein-coupled rhodopsin-type receptor on

the surface of target cells. [1] PGE2 was produced during inflammatory responses and it

suppressed the production of cytokines and chemokines induced by lipopolysaccharide

(LPS)-stimulated macrophages [2, 3] and dendritic cells. [4] Elsewhere we reported that

PGE2 modulates the expression of polyI:C-induced pro-inflammatory genes in human

conjunctival epithelial cells. [5]

There are four PGE receptor subtypes, EP1, EP2, EP3, and EP4. Intestinal

epithelium has been reported to express EP4 mRNA, [6] and intestinal homeostasis was

maintained- and the immune response was down-regulated by EP4. [7] We documented

that while normal human conjunctival epithelium expressed EP4 protein, it was

down-regulated in devastating ocular surface inflammatory disorders such as chronic

Stevens-Johnson syndrome (SJS)/Toxic epidermal necrolysis (TEN) and chronic ocular

cicatricial pemphigoid (OCP). [8] Here we examine the expression of EP4 in conjunctival

epithelium of patients with ocular surface disorders such as chemical eye burn, Mooren’s

ulcer, severe graft versus host disease (GVHD), and of patients in the subacute stage of

SJS/TEN.

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Materials and Methods

Human conjunctival tissues

This study was approved by the Institutional Review Board of Kyoto Prefectural

University of Medicine, Kyoto, Japan. All experiments were conducted in accordance

with the principles set forth in the Helsinki Declaration.

The controls for immunohistochemical analyses were nearly normal conjunctival

tissues obtained during surgery for conjunctivochalasis. We also prepared human

conjunctival tissues from samples obtained during surgery to reconstruct the ocular

surface in 3 patients with chemical eye burn, 2 patients with sub-acute SJS/TEN, one

patient with severe GVHD, and from 4 patients with Mooren's ulcer undergoing resection

of inflammatory conjunctiva.

For quantitative RT-PCR the controls were nearly normal conjunctival tissues

obtained at surgery for conjunctivochalasis. We also prepared human conjunctival tissues

from samples obtained during surgery to reconstruct the ocular surface in 4 patients in the

chronic stage of SJS/TEN and 4 OCP patients in the chronic stage.

Immunohistochemistry

For EP4 staining we used rabbit polyclonal antibody to EP4 (Cayman Chemical

Co., Ann Arbor, MI). The secondary antibody (Biotin-SP-conjugated AffiniPure F(ab’)2

fragment donkey anti-rabbit IgG (H+L), 1:500 dilution; Jackson Immuno Research,

Baltimore, MD) was applied for 30 min. The VECTASTAIN ABC reagent (Vector

Laboratories, Inc., Burlingame, CA) was used for increased sensitivity with peroxidase

substrate solution (DAB substrate kit; Vector) as a chromogenic substrate.

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Quantitative RT-PCR

Total RNA was isolated from conjunctival tissue sections using the RNeasy mini

kit (Qiagen) according to the manufacturer’s instructions. The RT reaction was with the

SuperScriptTM

preamplification kit (Invitrogen). Quantitative RT-PCR was on an

ABI-prism 7700 instrument (Applied Biosystems, Foster City, CA). The probes for

human PTGER4 and human GAPDH were from Applied Biosystems. For cDNA

amplification we performed PCR in a 25-µl total volume that contained a 1 µl cDNA

template in 2 × TaqMan universal PCR master mix (Applied Biosystems) at 50°C for 2

min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min.

The results were analyzed with sequence detection software (Applied Biosystems). The

quantification data were normalized to the expression of the housekeeping gene GAPDH.

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Results

EP4 protein was detected in nearly normal conjunctival epithelium from patients

with conjunctivochalasis (Fig. 1A) and in conjunctival tissues from 3 patients with

chemical eye burn (Fig. 1B). Its expression varied in conjunctival epithelium from 4

patients with Mooren’s ulcer (Fig. 1C): in one patient is was similar to the control, in 2

patients it was slightly lower than in the control, and in the remaining patient it was not

detected. There was no EP4 immunoreactivity in conjunctival epithelium from 2 patients

with subacute SJS/TEN (Fig. 1D), nor from patients with severe GVHD (Fig. 1E), as

same as chronic SJS/TEN, or OCP. [8]

We found that, as in normal human conjunctival epithelium, EP4 is expressed in

conjunctival epithelium from patients with chemical eye burn. On the other hand, it was

strongly down-regulated in conjunctival epithelium from patients with SJS/TEN, OCP, or

severe GVHD.

To confirm the down-regulation of EP4 in the ocular surface of SJS and OCP

patients we examined the expression of PTGER4 mRNA in control conjunctival tissues

from 6 conjunctival chalasis patients and in conjunctival tissues from 4 SJS/TEN- and 4

OCP patients. Representative findings of EP4 immunoreactivity in each of these groups

are shown in Fig. 2A. Although EP4 protein was detected in tissues from patients with

conjunctivochalasis (controls), conjunctival epithelium from SJS- and OCP patients did

not manifest EP4 immunoreactivity. PTGER4 mRNA was significantly lower in

conjunctival tissues from SJS and OCP patients than in the control conjunctivochalasis

samples (Fig. 2B).

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Discussion

Elsewhere we reported the expression of EP4 in normal human conjunctival

epithelium and its down-regulation in conjunctival epithelium from patients with

SJS/TEN and OCP. [8] Here we document that EP4 is expressed normally in conjunctival

epithelium from patients with severe chemical eye burn which, like SJS/TEN and OCP, is

a devastating ocular surface disorder. We also confirmed that in conjunctival tissues from

SJS/TEN and OCP patients its mRNA expression was significantly down-regulated.

On the ocular surface of patients with severe chemical eye burn, conjunctival

invasion into the cornea may occur due to the stem cell deficiency of corneal epithelial

cells. This results in devastating ocular surface disorders similar to OCP and SJS/TEN.

However, in the conjunctiva of patients with severe chemical eye burns, EP4 expression

was not down-regulated.

In patients with Mooren’s ulcer, an ocular surface inflammatory disease, the

expression of EP4 protein varied; in some patients it was down-regulated. In patients in

the sub-acute stage of SJS/TEN with ocular surface inflammation, the expression of EP4

protein was remarkably down-regulated.

Kabashima et al. [7] reported that in mice, EP4 deficiency impaired mucosal

barrier function and induced the aggregation of lymphocytes and neutrophils in the colon,

and that the administration of an EP4-selective agonist to wild-type mice ameliorated

severe colitis. In mice treated with an EP4-selective antagonist the recovery from colitis

was suppressed, leading them to conclude that EP4 maintains intestinal homeostasis by

preserving mucosal integrity and down-regulating the immune response. On the other

hand, Yao et al. [9] found that PGE2 acting on its receptor EP4 on T- and dendritic cells

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not only facilitated TH1 cell differentiation but also amplified interleukin-23-mediated

TH17 cell expansion in vitro. The administration of an EP4-selective antagonist to mice

with experimental autoimmune encephalomyelitis or contact hypersensitivity decreased

the accumulation of both TH1 and TH17 cells in regional lymph nodes and suppressed

disease progression. Based on these observations they concluded that PGE2-EP4

signaling promotes immune inflammation.

In human conjunctival tissues EP4 protein was expressed in epithelial cells but

not in cells infiltrating subconjunctival tissues. We posit that the down-regulation of EP4

in conjunctival epithelium is associated with the ocular surface inflammation seen in

patients with OCP, SJS/TEN, and Mooren’s ulcer because as in the acute or sub-acute

stage, patients in the chronic phase of OCP and SJS/TEN manifested mucosal

inflammation on the ocular surface.

On the other hand, elsewhere we reported that although EP3 and EP2 agonists

suppressed the production of CCL5, CXCL11, and CCL20 in response to polyI:C

stimulation, these chemokines were not suppressed by the EP4 agonist in human

conjunctival epithelial cells. [5] Studies are underway in our laboratory to elucidate the

function of EP4 in conjunctival epithelial cells.

In summary, EP4 is expressed not only in normal conjunctival epithelium but also

in conjunctival epithelium from patients with chemical eye burns and some patients with

Mooren’s ulcer. On the other hand, it is strongly down-regulated in conjunctival

epithelium from patients with OCP and chronic- and subacute SJS/TEN.

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Figure Legends

Figure 1

Immunohistological analysis of prostaglandin E receptor subtype EP4 in conjunctival

epithelium of patients with ocular surface diseases.

A. Nearly normal conjunctival tissues from patients with conjunctivochalasis.

B. Conjunctival tissues of chemical eye burn patients in need of ocular surface

reconstruction.

C. Inflammatory conjunctival tissues of patients with active Mooren’s ulcer

requiring resection of the inflammatory conjunctiva.

D. Conjunctival tissues of SJS/TEN patients in the sub-acute stage.

E. Conjunctival tissues of a patient with severe GVHD.

Each scale bar represents 100 µm.

Figure 2

The expression of PTGER4 mRNA in conjunctival tissues from patients with SJS/TEN,

OCP and the controls.

A. Representative findings of EP4 immunoreactivity of each group (control,

SJS/TEN, OCP).

B. Expression of PTGER4 mRNA in human conjunctival tissues (*p < 0.05).

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References

1. Matsuoka T, Narumiya S: Prostaglandin receptor signaling in disease.

ScientificWorldJournal 2007, 7:1329-1347.

2. Takayama K, Garcia-Cardena G, Sukhova GK, et al.: Prostaglandin E2

suppresses chemokine production in human macrophages through the EP4

receptor. J Biol Chem 2002, 277:44147-44154.

3. Xu XJ, Reichner JS, Mastrofrancesco B, et al.: Prostaglandin E2 suppresses

lipopolysaccharide-stimulated IFN-beta production. J Immunol 2008,

180:2125-2131.

4. Shiraishi H, Yoshida H, Saeki K, et al: Prostaglandin E2 is a major soluble

factor produced by stromal cells for preventing inflammatory cytokine

production from dendritic cells. Int Immunol 2008, 20:1219-1229.

5. Ueta M, Matsuoka T, Yokoi N, et al: Prostaglandin E2 suppresses

polyinosine-polycytidylic acid (polyI:C)-stimulated cytokine production via

prostaglandin E2 receptor (EP) 2 and 3 in human conjunctival epithelial

cells. Br J Ophthalmol 2011, 95:859-863.

6. Morimoto K, Sugimoto Y, Katsuyama M, et al: Cellular localization of

mRNAs for prostaglandin E receptor subtypes in mouse gastrointestinal

tract. Am J Physiol 1997, 272:G681-687.

7. Kabashima K, Saji T, Murata T, et al: The prostaglandin receptor EP4

suppresses colitis, mucosal damage and CD4 cell activation in the gut. J Clin

Invest 2002, 109:883-893.

8. Ueta M, Sotozono C, Yokoi N, et al: Prostaglandin E receptor 4 expression in

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human conjunctival epithelium and its downregulation in devastating

ocular surface inflammatory disorders. Arch Ophthalmol 2010,

128(10):1369-1371.

9. Yao C, Sakata D, Esaki Y, et al: Prostaglandin E2-EP4 signaling promotes

immune inflammation through Th1 cell differentiation and Th17 cell

expansion. Nat Med 2009, 15:633-640.

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ACKNOWLEDGMENTS

We thank Chikako Endo for technical assistance. This work was supported in part

by grants-in-aid for scientific research from the Japanese Ministry of Health, Labour and

Welfare, the Japanese Ministry of Education, Culture, Sports, Science and Technology,

CREST from JST, a research grant from the Kyoto Foundation for the Promotion of

Medical Science, the Intramural Research Fund of Kyoto Prefectural University of

Medicine, and an Immunological Research Grant from the Shimizu Foundation.

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Immunohistological analysis of prostaglandin E receptor subtype EP4 in conjunctival epithelium of patients with ocular surface diseases. 53x53mm (300 x 300 DPI)

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40x45mm (300 x 300 DPI)

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STROBE 2007 (v4) Statement—Checklist of items that should be included in reports of case-control studies

Section/Topic Item

# Recommendation

Reported on

page #

Title and abstract 1 (a) Indicate the study’s design with a commonly used term in the title or the abstract １

(b) Provide in the abstract an informative and balanced summary of what was done and what was found ３

Introduction

Background/rationale 2 Explain the scientific background and rationale for the investigation being reported ４

Objectives 3 State specific objectives, including any prespecified hypotheses ４

Methods

Study design 4 Present key elements of study design early in the paper ５

Setting 5 Describe the setting, locations, and relevant dates, including periods of recruitment, exposure, follow-up, and data collection ５

Participants 6 (a) Give the eligibility criteria, and the sources and methods of case ascertainment and control selection. Give the rationale for

the choice of cases and controls

５

(b) For matched studies, give matching criteria and the number of controls per case

Variables 7 Clearly define all outcomes, exposures, predictors, potential confounders, and effect modifiers. Give diagnostic criteria, if

applicable

５

Data sources/

measurement

8* For each variable of interest, give sources of data and details of methods of assessment (measurement). Describe comparability

of assessment methods if there is more than one group

５

Bias 9 Describe any efforts to address potential sources of bias

Study size 10 Explain how the study size was arrived at

Quantitative variables 11 Explain how quantitative variables were handled in the analyses. If applicable, describe which groupings were chosen and why ５

Statistical methods 12 (a) Describe all statistical methods, including those used to control for confounding ６

(b) Describe any methods used to examine subgroups and interactions

(c) Explain how missing data were addressed

(d) If applicable, explain how matching of cases and controls was addressed

(e) Describe any sensitivity analyses

Results

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Participants 13* (a) Report numbers of individuals at each stage of study—eg numbers potentially eligible, examined for eligibility, confirmed

eligible, included in the study, completing follow-up, and analysed

(b) Give reasons for non-participation at each stage

(c) Consider use of a flow diagram

Descriptive data 14* (a) Give characteristics of study participants (eg demographic, clinical, social) and information on exposures and potential

confounders

７

(b) Indicate number of participants with missing data for each variable of interest

Outcome data 15* Report numbers in each exposure category, or summary measures of exposure

Main results 16 (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their precision (eg, 95% confidence

interval). Make clear which confounders were adjusted for and why they were included

(b) Report category boundaries when continuous variables were categorized

(c) If relevant, consider translating estimates of relative risk into absolute risk for a meaningful time period

Other analyses 17 Report other analyses done—eg analyses of subgroups and interactions, and sensitivity analyses

Discussion

Key results 18 Summarise key results with reference to study objectives ７

Limitations 19 Discuss limitations of the study, taking into account sources of potential bias or imprecision.

Discuss both direction and magnitude of any potential bias

Interpretation 20 Give a cautious overall interpretation of results considering objectives, limitations, multiplicity of analyses, results from similar

studies, and other relevant evidence

Generalisability 21 Discuss the generalisability (external validity) of the study results ８

Other information

Funding 22 Give the source of funding and the role of the funders for the present study and, if applicable, for the original study on which the

present article is based

１３

*Give information separately for cases and controls in case-control studies and, if applicable, for exposed and unexposed groups in cohort and cross-sectional studies.

Note: An Explanation and Elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting. The STROBE

checklist is best used in conjunction with this article (freely available on the Web sites of PLoS Medicine at http://www.plosmedicine.org/, Annals of Internal Medicine at

http://www.annals.org/, and Epidemiology at http://www.epidem.com/). Information on the STROBE Initiative is available at www.strobe-statement.org.

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disorders

Journal: BMJ Open

Manuscript ID: bmjopen-2012-001330.R1


Date Submitted by the Author: 28-Jun-2012




Ophthalmology

Secondary Subject Heading: Ophthalmology






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Section/Topic Item

# Recommendation

Reported on

page #



Introduction



Methods





５



applicable

５

Data sources/

measurement



５









Results

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confounders

７








Discussion







Other information



１３





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patients with ocular surface disorders: Case-control study



a



a


Kyoto,

Japan










Telephone: 81-75-251-5578

Fax: 81-75-251-5663


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Key words



Abbreviations




GVHD: Graft versus host disease

OCP: Ocular cicatricial pemphigoid


PG: Prostaglandin

1) Substantial contributions to conception and design, acquisition of data, or analysis

and interpretation of data: Mayumi Ueta, Chie Sotozono, Keiko Yamada,

Norihiko Yokoi, Tsutomu Inatomi, Shigeru Kinoshita

2) Drafting the article or revising it critically for important intellectual content:

Mayumi Ueta, Chie Sotozono, Keiko Yamada, Norihiko Yokoi, Tsutomu Inatomi,

Shigeru Kinoshita

3) Final approval of the version to be published; Mayumi Ueta, Chie Sotozono,

Keiko Yamada, Norihiko Yokoi, Tsutomu Inatomi, Shigeru Kinoshita

No additional data are available.

Competing interest statement: None

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Abstract

Objectives: To confirm the down-regulation of PTGER4 mRNA in the conjunctiva of

SJS/TEN and OCP patients and to examine the expression of its EP4 protein in the

conjunctival epithelium of patients with various ocular surface disorders.

Design: Case-control study

Setting & Participants: We performed quantitative RT-PCR analysis of EP4 in

conjunctival tissue sections from patients with SJS/TEN and OCP to confirm the

down-regulation of EP4 mRNA expression. We also analyzed EP4 immunohistologically

in other ocular surface disorders. Conjunctival tissues were obtained from patients

undergoing surgical reconstruction of the ocular surface due to chemical eye burns,

sub-acute- or chronic SJS/TEN, chronic ocular cicatricial pemphigoid (OCP), severe


resection of the inflammatory conjunctiva.

Primary and secondary outcome measures: The expression of EP4 mRNA and EP4

protein assessed by quantitative RT-PCR assay and immunohistological methods.

Results: PTGER4 mRNA was significantly lower in conjunctival tissues from SJS and

OCP patients than in the control conjunctivochalasis samples. EP4 protein was detected

in conjunctival epithelium from patients with chemical eye burn and in control

conjunctival epithelium from patients with conjunctivochalasis. Its expression varied in

conjunctival epithelium from patients with Mooren’s ulcer. We did not detect EP4

immunoreactivity in conjunctival epithelium from patients with subacute SJS/TEN,

severe GVHD, chronic SJS/TEN, or OCP.



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Introduction




the surface of target cells [1]. PGE2 is produced during inflammatory responses and it

suppresses the production of cytokines and chemokines induced by lipopolysaccharide

(LPS)-stimulated macrophages [2, 3] and dendritic cells [4]. Elsewhere we reported that


conjunctival epithelial cells [5].

There are four PGE receptor subtypes, EP1, EP2, EP3, and EP4. The intestinal

epithelium has been reported to express EP4 mRNA [6] and intestinal homeostasis was

maintained and the immune response down-regulated by EP4 [7]. We documented that

while normal human conjunctival epithelium expressed EP4 protein, it was


Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and chronic ocular

cicatricial pemphigoid (OCP) [8]. Here we examined the mRNA expression of EP4 in the

conjunctiva of SJS/TEN and OCP patients in the chronic stage to confirm that EP4 is

down-regulated in their conjunctiva. We also examined the expression of EP4 protein in

the conjunctival epithelium of patients with various ocular surface disorders such as

chemical eye burn, Mooren’s ulcer, severe graft versus host disease (GVHD), and of

patients in the sub-acute stage of SJS/TEN.

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chronic stage of SJS/TEN and 4 patients in the chronic stage of OCP.


tissues obtained during surgery for conjunctivochalasis, a disease in which the

conjunctiva relaxes due to aging, resulting in a foreign body sensation on the ocular

surface. We also prepared human conjunctival tissues from samples obtained during

surgery to reconstruct the ocular surface in 3 patients with chemical (alkali) eye burn (2 in

the chronic- and one in the sub-acute stage), 2 patients with sub-acute SJS/TEN, one


of inflammatory conjunctiva. SJS/TEN, OCP, Mooren's ulcer, chemical burn, and GVHD

are all ocular surface inflammatory diseases with persistent inflammation on the ocular

surface not only in the acute- but also in the chronic stage.

Quantitative RT-PCR



SuperScriptTM


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amplification we performed PCR in a 25-µl total volume that contained a 1-µl cDNA












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7

Results





are shown in Fig. 1A. Although EP4 protein was detected in the control tissues,

conjunctival epithelium from SJS- and OCP patients did not manifest EP4

immunoreactivity. PTGER4 mRNA was significantly lower in conjunctival tissues from

SJS and OCP patients than in the control conjunctivochalasis samples (Fig. 1B).




patients with Mooren’s ulcer (Fig. 2C): in one patient is was similar to the control, in 2 it

was slightly lower than in the control, and in the remaining patient it was not detected.

There was no EP4 immunoreactivity in conjunctival epithelium from 2 patients with

subacute SJS/TEN (Fig. 2D), a patient with severe GVHD (Fig. 2E), and patients with

chronic SJS/TEN or OCP [8].


conjunctival epithelium from patients with chemical eye burn. On the other hand, EP4

immunoreactivity was not detected in conjunctival epithelium from patients with

SJS/TEN, OCP, or severe GVHD. We did not detect EP4 protein in cells infiltrating

subconjunctival tissues in any of the human conjunctival tissues we examined.

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8

Discussion



SJS/TEN and OCP [8]. Here we confirmed that in conjunctival tissues from SJS/TEN and

OCP patients its mRNA expression was significantly down-regulated, and we also

document that EP4 is expressed normally in conjunctival epithelium from patients with

severe chemical eye burn which, like SJS/TEN and OCP, is a devastating ocular surface

disorder.

















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patients with OCP, SJS/TEN, and Mooren’s ulcer.




conjunctival epithelial cells [5]. Studies are underway in our laboratory to elucidate the






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Figure Legends

Figure 1



A. Representative findings of EP4 immunoreactivity in each group (control,

SJS/TEN, OCP).


Figure 2




B. Conjunctival tissues from patients with chemical eye burn requiring ocular

surface reconstruction.

C. Inflammatory conjunctival tissues from patients with active Mooren’s ulcer


D. Conjunctival tissues from SJS/TEN patients in the sub-acute stage.

E. Conjunctival tissues from a patient with severe GVHD.


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References








180:2125-2131.













Invest 2002, 109:883-893.



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128(10):1369-1371.




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ACKNOWLEDGMENTS







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1





a



a


Kyoto,

Japan










Telephone: 81-75-251-5578

Fax: 81-75-251-5663


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2

Key words



Abbreviations







PG: Prostaglandin






Shigeru Kinoshita





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Abstract





Setting & Participants: Conjunctival tissues were obtained from patients undergoing

surgical reconstruction of the ocular surface due to chemical eye burns, sub-acute and

chronic stage SJS/TEN, chronic stage ocular cicatricial pemphigoid (OCP) or severe


resection of the inflammatory conjunctiva. We performed quantitative RT-PCR analysis

immunohistological analysis of EP4 and quantitative RT-PCR analysis of in conjunctival

tissue sections from patients with SJS/TEN and OCP to confirm the down-regulation of

EP4 mRNA expression. We also analyzed EP4 immunohistologically in other ocular

surface disorders. Conjunctival tissues were obtained from patients undergoing surgical

reconstruction of the ocular surface due to chemical eye burns, sub-acute- or chronic

SJS/TEN, chronic ocular cicatricial pemphigoid (OCP), severe graft versus host disease

(GVHD), and from patients with Mooren's ulcers treated by resection of the

inflammatory conjunctiva.

Primary and secondary outcome measures: The expression of EP4 mRNA and

expression of EP4 protein assessed by quantitative RT-PCR assay and

immunohistological methods. and expression of EP4 mRNA.




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Introduction











maintained and the immune response down-regulated by EP4 [7]. We documented that

while normal human conjunctival epithelium expressed EP4 protein, it was


Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and chronic ocular

cicatricial pemphigoid (OCP) [8]. Here we examined the mRNA expression of EP4 in the

conjunctiva of SJS/TEN and OCP patients in the chronic stage to confirm that EP4 is

down-regulated in their conjunctiva. We also examined the expression of EP4 protein in

the conjunctival epithelium of patients with various ocular surface disorders such as

chemical eye burn, Mooren’s ulcer, severe graft versus host disease (GVHD), and of

patients in the sub-acute stage of SJS/TEN.

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Quantitative RT-PCR



SuperScriptTM


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Quantitative RT-PCR



SuperScriptTM




amplification we performed PCR in a 25-µl total volume that contained a 1 µl cDNA


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Results





are shown in Fig. 1A. Although EP4 protein was detected in the control tissues,

conjunctival epithelium from SJS- and OCP patients did not manifest EP4

immunoreactivity. PTGER4 mRNA was significantly lower in conjunctival tissues from

SJS and OCP patients than in the control conjunctivochalasis samples (Fig. 1B).




patients with Mooren’s ulcer (Fig. 2C): in one patient is was similar to the control, in 2 it

was slightly lower than in the control, and in the remaining patient it was not detected.

There was no EP4 immunoreactivity in conjunctival epithelium from 2 patients with

subacute SJS/TEN (Fig. 2D), a patient with severe GVHD (Fig. 2E), and patients with

chronic SJS/TEN or OCP [8].



immunoreactivity was not detected it was strongly down-regulated in conjunctival

epithelium from patients with SJS/TEN, OCP, or severe GVHD. We did not detect EP4

protein in cells infiltrating subconjunctival tissues in any of the human conjunctival

tissues we examined.


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are shown in Fig. 2A. Although EP4 protein was detected in tissues from patients with

conjunctivochalasis (controls), conjunctival epithelium from SJS- and OCP patients did

not manifest EP4 immunoreactivity. PTGER4 mRNA was significantly lower in

conjunctival tissues from SJS and OCP patients than in the control conjunctivochalasis

samples (Fig. 2B).

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11

Discussion







disorder. We also confirmed that in conjunctival tissues from SJS/TEN and OCP patients

its mRNA expression was significantly down-regulated.
















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patients with OCP, SJS/TEN, and Mooren’s ulcer because as in the acute or sub-acute

stage, patients in the chronic phase of OCP and SJS/TEN manifested mucosal

inflammation on the ocular surface.










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Figure Legends

Figure 1











Figure 2

Figure 1




SJS/TEN, OCP).


Figure 1

Figure 2

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F. Nearly normal conjunctival tissues from patients with conjunctivochalasis.

G. Conjunctival tissues from patients with chemical eye burn requiring ocular


H. Inflammatory conjunctival tissues from patients with active Mooren’s ulcer


I. Conjunctival tissues from SJS/TEN patients in the sub-acute stage.

J. Conjunctival tissues from a patient with severe GVHD.


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15

References








180:2125-2131.













Invest 2002, 109:883-893.



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128(10):1369-1371.




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ACKNOWLEDGMENTS







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190x275mm (300 x 300 DPI)

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190x275mm (300 x 300 DPI)

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disorders

Journal: BMJ Open

Manuscript ID: bmjopen-2012-001330.R2


Date Submitted by the Author: 28-Jul-2012




Ophthalmology

Secondary Subject Heading: Genetics and genomics






jopen.bmj.com

/B

MJ O


ctober 2012. Dow

nloaded from




Section/Topic Item

# Recommendation

Reported on

page #



Introduction



Methods





５



applicable

５

Data sources/

measurement



５









Results

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confounders

７








Discussion







Other information



１３





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1





a



a


Kyoto,

Japan










Telephone: 81-75-251-5578

Fax: 81-75-251-5663


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Key words



Abbreviations







PG: Prostaglandin






Shigeru Kinoshita





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Abstract
























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4

Introduction











maintained and the immune response down-regulated by EP4 [7]. The ocular surface is

also one of the mucosa that is in contact with commensal bacteria like the intestine.

Therefore, we focused the expression of EP4 in human conjunctival epithelium and the

difference of its expression between various ocular surface diseases.

We documented that while normal human conjunctival epithelium expressed EP4

protein, it was down-regulated in devastating ocular surface inflammatory disorders such

as chronic Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and

chronic ocular cicatricial pemphigoid (OCP) [8]. Here we examined the mRNA

expression of EP4 in the conjunctiva of SJS/TEN and OCP patients in the chronic stage to

confirm that EP4 is down-regulated in their conjunctiva. We also examined the

expression of EP4 protein in the conjunctival epithelium of patients with various ocular

surface disorders such as chemical eye burn, Mooren’s ulcer, severe graft versus host

disease (GVHD), and of patients in the sub-acute stage of SJS/TEN.

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Quantitative RT-PCR



SuperScriptTM


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Data analysis

Data were expressed as the mean ± SEM and evaluated by the Student’s t-test using the

Microsoft Excel software program.

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7

Results

We previously documented that EP4 protein expression was down-regulated in

conjunctival epithelium of devastating ocular surface inflammatory disorders such as

chronic SJS/TEN and chronic OCP [8]. In this study, to confirm the down-regulation of

EP4 in the ocular surface of SJS/TEN and OCP patients we examined the expression of

PTGER4 mRNA in control conjunctival tissues from 6 conjunctival chalasis patients and

in conjunctival tissues from 4 SJS/TEN- and 4 OCP patients. Representative findings of

EP4 immunoreactivity in each of these groups are shown in Fig. 1A. Although EP4

protein was detected in the control tissues, conjunctival epithelium from SJS- and OCP

patients did not manifest EP4 immunoreactivity. PTGER4 mRNA was significantly

lower in conjunctival tissues from SJS/TEN and OCP patients than in the control

conjunctivochalasis samples (Fig. 1B).

Moreover, we examined the expression of EP4 protein in the conjunctival

epithelium of patients with other various ocular surface disorders. EP4 protein was

detected in nearly normal conjunctival epithelium from patients with conjunctivochalasis

(Fig. 2A) and in conjunctival tissues from 3 patients with chemical eye burn (Fig. 2B). Its

expression varied in conjunctival epithelium from 4 patients with Mooren’s ulcer (Fig.

2C): in one patient is was similar to the control, in 2 it was slightly lower than in the

control, and in the remaining patient it was not detected. There was no EP4

immunoreactivity in conjunctival epithelium from 2 patients with subacute SJS/TEN (Fig.

2D), a patient with severe GVHD (Fig. 2E) as same as patients with chronic SJS/TEN or

OCP [8].



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Discussion







disorder.










Our results suggest that it is possible that EP4 in conjunctival epithelium might

contribute the ocular surface homeostasis, while the EP4 may not necessarily be

down-regulated in all devastating ocular surface disorders.





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Figure Legends

Figure 1




SJS/TEN, OCP).


Figure 2











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12

References








180:2125-2131.













Invest 2002, 109:883-893.



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128(10):1369-1371.




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ACKNOWLEDGMENTS







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1





a



a


Kyoto,

Japan










Telephone: 81-75-251-5578

Fax: 81-75-251-5663


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2

Key words



Abbreviations







PG: Prostaglandin






Shigeru Kinoshita





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Abstract
























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4

Introduction











maintained and the immune response down-regulated by EP4 [7]. The ocular surface is

also one of the mucosa that is in contact with commensal bacteria like the intestine.

Therefore, we focused the expression of EP4 in human conjunctival epithelium and the

difference of its expression between various ocular surface diseases.

We documented that while normal human conjunctival epithelium expressed EP4

protein, it was down-regulated in devastating ocular surface inflammatory disorders such

as chronic Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and

chronic ocular cicatricial pemphigoid (OCP) [8]. Here we examined the mRNA

expression of EP4 in the conjunctiva of SJS/TEN and OCP patients in the chronic stage to

confirm that EP4 is down-regulated in their conjunctiva. We also examined the

expression of EP4 protein in the conjunctival epithelium of patients with various ocular

surface disorders such as chemical eye burn, Mooren’s ulcer, severe graft versus host

disease (GVHD), and of patients in the sub-acute stage of SJS/TEN.

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5




















Quantitative RT-PCR



SuperScriptTM


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6















Data analysis

Data were expressed as the mean ± SEM and evaluated by the Student’s t-test using the

Microsoft Excel software program.

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7

Results

We previously documented that EP4 protein expression was down-regulated in

conjunctival epithelium of devastating ocular surface inflammatory disorders such as

chronic SJS/TEN and chronic OCP [8]. In this study, to confirm the down-regulation of

EP4 in the ocular surface of SJS/TEN and OCP patients we examined the expression of

PTGER4 mRNA in control conjunctival tissues from 6 conjunctival chalasis patients and

in conjunctival tissues from 4 SJS/TEN- and 4 OCP patients. Representative findings of

EP4 immunoreactivity in each of these groups are shown in Fig. 1A. Although EP4

protein was detected in the control tissues, conjunctival epithelium from SJS- and OCP

patients did not manifest EP4 immunoreactivity. PTGER4 mRNA was significantly

lower in conjunctival tissues from SJS/TEN and OCP patients than in the control

conjunctivochalasis samples (Fig. 1B).

Moreover, we examined the expression of EP4 protein in the conjunctival

epithelium of patients with other various ocular surface disorders. EP4 protein was

detected in nearly normal conjunctival epithelium from patients with conjunctivochalasis

(Fig. 2A) and in conjunctival tissues from 3 patients with chemical eye burn (Fig. 2B). Its

expression varied in conjunctival epithelium from 4 patients with Mooren’s ulcer (Fig.

2C): in one patient is was similar to the control, in 2 it was slightly lower than in the

control, and in the remaining patient it was not detected. There was no EP4

immunoreactivity in conjunctival epithelium from 2 patients with subacute SJS/TEN (Fig.

2D), a patient with severe GVHD (Fig. 2E) as same as patients with chronic SJS/TEN or

OCP [8].



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Discussion







disorder.










Our results suggest that it is possible that EP4 in conjunctival epithelium might

contribute the ocular surface homeostasis, while the EP4 could not necessarily be

down-regulated in all devastating ocular surface disorders.





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Figure Legends

Figure 1




SJS/TEN, OCP).


Figure 2











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References








180:2125-2131.













Invest 2002, 109:883-893.



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128(10):1369-1371.




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ACKNOWLEDGMENTS







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190x275mm (300 x 300 DPI)

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